Cyclodextrin conjugates

Abstract

It has been discovered that the uptake of anionic charged species into cells can be enhanced by noncovalently associating such species with specifically modified forms of cyclodextrin. The invention modified forms of cyclodextrin form well defined stoichiometric complexes with anionic charged molecules. This discovery enables one to produce various compositions containing anionic charged molecules and facilitates methods for enhancing the cellular uptake of double-stranded or hairpin nucleic acid.

1 . A construct represented by formula I:
   CA 1 -L 1 -CD-L 2 -CA 2    (1)   
       wherein,
 CD=cyclodextrin; 
 L 1 , L 2 =linker; and 
 CA 1 , CA 2 =cationic arm. 
 
     
     
         2 . The construct of  claim 1 , wherein said cyclodextrin is alpha, beta or gamma cyclodextrin. 
     
     
         3 . The construct of  claim 2 , further comprising a bio-recognition molecule. 
     
     
         4 . The construct of  claim 1 , wherein each linker is independently selected from the group consisting of a covalent bond, a disulfide linkage, a protected disulfide linkage, an ether linkage, a thioether linkage, a sulfoxide linkage, a sulfonate linkage, an ester linkage, an amide linkage, a carbamate linkage, a dithiocarbamate linkage, an amine linkage, a hydrazone linkage, a sulfonamide linkage, an urea linkage, and combinations thereof 
     
     
         5 . The construct of  claim 4 , wherein each linker is covalently linked to the 6-position of A,D-rings, A,C-rings or A,E-rings of said cyclodextrin. 
     
     
         6 . The construct of  claim 1 , wherein each cationic arm comprises a plurality of residues selected from amines, guanidines, amidines, N-containing heterocycles, or combinations thereof 
     
     
         7 . The construct of  claim 1 , wherein each cationic arm comprises a plurality of reactive units selected from the group consisting of alpha-amino acids, beta-amino acids, gamma-amino acids, cationically functionalized monosaccharides, cationically functionalized ethylene glycols, ethylene imines, substituted ethylene imines, N-substituted spermine, N-substituted spermidine, and combinations thereof. 
     
     
         8 . The construct of  claim 7 , wherein each cationic arm comprises an oligomer independently selected from the group consisting of oligopeptide, oligoamide, cationically functionalized oligoether, cationically functionalized oligosaccharide, oligoamine, oligoethyleneimine, and combinations thereof 
     
     
         9 . The construct of  claim 8 , wherein said oligomer is an oligopeptide. 
     
     
         10 . The construct of  claim 9 , wherein substantially all of the amino acid residues of said oligopeptide are capable of forming positive charges. 
     
     
         11 . The construct of  claim 10 , wherein said oligopeptide comprises 3 to 50 amino acids. 
     
     
         12 . The construct of  claim 1 , wherein
 CD=beta-cyclodextrin;   L 1 , L 2 =linker; and   CA 1 , CA 2  comprise independently an oligopeptide;   wherein each linker is covalently linked to the 6-position of A,D-rings of said beta-cyclodextrin.   
     
     
         13 . A complex comprising a construct of  claim 1  associated with an anionic charged molecule. 
     
     
         14 . The complex of  claim 13 , wherein said anionic charged molecule is selected from the group consisting of a double-stranded nucleic acid, hairpin nucleic acid, single-stranded DNA, double-stranded DNA, single-stranded RNA, double-stranded RNA, and oligonucleotide comprising non-natural monomers. 
     
     
         15 . A composition comprising a pharmaceutical excipient, an anionic charged molecule and a construct of  claim 1 , or a pharmaceutically acceptable ester, salt, or hydrate thereof. 
     
     
         16 . A method of attenuating expression of a target gene in treated cells comprising delivering a construct of  claim 1  and a double-stranded or hairpin nucleic acid to said cell. 
     
     
         17 . A method for delivering an anionic charged molecule to a cell, said method comprising:
 a) binding non-covalently a construct of  claim 1  to said anionic charged molecule to form a complex; and   b) contacting said cell with said complex; wherein said anionic charged molecule is taken up by said cell.   
     
     
         18 . A method for delivering an anionic charged molecule to a cell, said method comprising contacting said cell with a complex prepared by binding non-covalently a
 construct of  claim 1  to said anionic charged molecule, wherein said anionic charged molecule is taken up by said cell.   
     
     
         19 . A method for stabilizing an anionic charged molecule in vivo or for reducing the susceptibility of an anionic charged molecule to self-aggregation, said method comprising contacting said anionic charged molecule with the construct of  claim 1 . 
     
     
         20 . A method for (a) increasing the temperature of hybrid dissociation of a double-stranded hairpin nucleic acid, (b) reducing the susceptibility of a double-stranded or hairpin nucleic acid to digestion by enzymatic nuclease or (c) reducing the susceptibility of a double-stranded or hairpin nucleic acid to hydrolysis of the phophodiester backbone, said method comprising contacting said nucleic acid with a construct of  claim 1 . 
     
     
         21 . A method for preparing a construct of formula I comprising:
 a) covalently attaching linkers L 1  and L 2  to a cyclodextrin; and   b) covalently attaching cationic arms CA 1  and CA 2  to L 1  and L 2 , respectively or   a′) covalently attaching a first linker to a first cationic arm to form L 1 -CA 1  and a second linker to a second arm to form L 2 -CA 2 ; and   b′) covalently attaching L 1 -CA 1  and L 2 -CA 2  to a cyclodextrin.   
     
     
         22 . A method comprising reacting an optionally substituted 6-perbenzyl cyclodextrin with a hydride reducing agent to produce a 6 A ,6 D  or a 6 A ,6 E  dihydroxyl cyclodextrin. 
     
     
         23 . The method  claim 22 , wherein said optionally substituted 6-perbenzyl cyclodextrin is 6-per-(p-methoxylbenzyl) cyclodextrin. 
     
     
         24 . The method  claim 23 , wherein said hydride reducing agent is an aluminum hydride reducing agent. 
     
     
         25 . The method of  claim 24 , wherein said aluminum hydride reducing agent is diisobutylaluminium hydride. 
     
     
         26 . A compound represented by formula II: 
       
         
           
           
               
               
           
         
       
       wherein:
 m is 0, 1 or 2; 
 p is 1 or 2, provided when p is 2, m is 1; 
 L 1  and L 2  are linkers independently selected from the group consisting of a covalent bond, a disulfide linkage, a protected disulfide linkage, an ether linkage, a thioether linkage, a sulfoxide linkage, a sulfonate linkage, an ester linkage, an amide linkage, a carbamate linkage, a dithiocarbamate linkage, an amine linkage, a hydrazone linkage, a sulfonamide linkage, an urea linkage, and combinations thereof; 
 R 1  is selected from the group consisting of hydrogen, alkyl, substituted alkyl, acyl, carbamoyl and silyl; 
 R 2  is selected from the group consisting of hydrogen, alkyl, substituted alkyl, and acyl; 
 X 1  and X 2  are independently displaceable functional groups; 
 with the proviso that R 1  and R2 are not the same; said ether linkage is not p-(allyloxy)phenyl ether linkage; and said amide linkage is not p-(allyloxy)benzoyl amide linkage.